Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria

Background Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. Results In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. Conclusion Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12934-024-02506-x.


Introduction
Non-conventional yeasts and bacteria are becoming so prevalent in the fields of biotechnology owing to their unique metabolic capabilities and ability to produce valuable compounds from inexpensive and renewable feedstocks [1,2].However, one of the challenges in using these organisms is the absence of appropriate promoters for regulating gene expression in these species.The majority of biotechnology promoters are obtained from model organisms, including Saccharomyces cerevisiae and Escherichia coli [3,4].
In the process of bioproduct production, selecting the proper host is a critical phase [5][6][7].Promising unconventional chassis cells include the yeasts Yarrowia lipolytica, Kluyveromyces, and Pichia pastoris, and the bacterium Corynebacterium glutamicum.Y. lipolytica is primarily used for the production of proteins, oils, terpenes, organic acids, and sugar alcohols due to the sufficient supply of acetyl-CoA, NADPH and the low glycosylation level of protein [8][9][10]; Kluyveromyces marxianus has shown significant effectiveness in producing aromatic chemicals and biofuel ethanol due to its favorable traits, including the ability to use a wide range of substrates, fast growth, and great resistance to high temperatures [11][12][13][14].Pichia pastoris is widely used in the production of heterologous proteins due to high protein secretion capacity and low glycosylation level.P. pastoris is also used as a one-carbon carbon source utilization chassis due to the natural methylotrophic characteristics [15].Corynebacterium glutamicum is widely used in the large-scale production of various L-amino acids, such as L-glutamate, L-lysine, L-serine, and L-threonine.C. glutamicum is also used to produce organic acids, biofuels, terpenoids and aromatic compounds [16,17].
A great challenge of bioproducts synthesis is the competition between cell native metabolism pathways and the heterologous target product synthesis pathways for limited cellular carbon resources.The dynamic metabolic engineering is an effective strategy for fine-tuning metabolic flux to maximize target product synthesis [18,19].In order to dynamically orchestrate the carbon flux, the heterologous synthesis pathways are often strengthened by engineering promoters and the competitive native pathways are generally altered by knocking out or knocking down [20][21][22][23][24].However, the competing pathways essential for normal cell growth cannot be completely removed.The dynamic up-regulation and down-regulation on multiple pathways simultaneously could be adjusted by promoter sets with diverse strengths [25][26][27].Promoters, the most basic transcriptional regulatory elements, have been used widely for gene expression and metabolic pathway engineering [28][29][30][31][32].The coordinated co-expression of multiple genes in multistep pathways is required for intricate synthetic biology.Multiple promoters are required for multistep metabolic pathways to avoid repeated usage of the same promoter at adjacent loci.The usage of the same promoter can result in genetic instability of engineered strains due to lost parts of the expression cassettes by loop-out homologous recombination [33,34].Replacing promoters with different sequences and regulatory strengths in the functional modules to adjust the adaptability of the chassis cells increases the output of the target products [35][36][37].There have been a number of interesting studies on metabolically designed microbial cell factories to generate bioproducts with different levels of promoters [38][39][40].For example, high-titer production of n-butanol from E. coli was achieved by using different expression levels of promoters [38].
Many promoters are incompatible in different hosts [41].The construction of pre-optimized chassis strains for biosynthesis pathways, promoter substitution, and redesigning, is always required in different hosts.Host specific promoters need to be selected to reconstruct biosynthetic pathways, which is a time-consuming and complicated construction process.Promoters with broad spectrum in different hosts are rare.The development of broad-spectrum promoters could enable synthetic circuit shuttles to be expressed between diverse host cells, from yeast to yeast, or even between the eukaryotic hosts and prokaryotic hosts [42,43].The feasibility of some heterologous yeast promoters in different expression systems have been characterized.For example, Kluyveromyces marxianus TPI and Hansenula polymorpha PMA promoters in P. pastoris [44], GAL1/2 promoters from other Saccharomyces species in S. cerevisiae [34], S. cerevisiae promoters (P GPD , P ADH , P TEF , and P CYC ) in K. marxianus [45] and the eukaryotic promoter GAL1/10 from S. cerevisiae direct expressing gene in E. coli [46].The development of promoters with broad host properties could enable rapid phenotyping of genetic constructs in different hosts.Therefore, the strength characterization of different promoters in different hosts is needed for multihost applications.
In this study, we aimed to find broad-spectrum promoter sets with different strengths to dynamically  balance the metabolic flux for the efficient production of high value-added bioproducts in different hosts.We selected five constitutive promoters of Y. lipolytica and K. marxianus respectively to compared promoter strength by the expression levels of reporter genes α-amylase (Oryza sativa, AMY1A, 1305 bp) [47] and red fluorescent protein (RFP, mRuby, JX489389.1771bp) [48] in different hosts.The broad-spectrum promoters with different strengths were characterized.Interestingly, we found two yeast promoters that could shuttle express reporter genes in E. coli, P. pastoris and C. glutamicum.These broadspectrum promoters will expand the synthetic biology toolbox and the application of bioengineering.

Strains, growth media, and culture conditions
The Y. lipolytica, K. marxianus, P. pastoris, E. coli and C. glutamicum strains are listed in Table 1.The thermotolerant Y. lipolytica CGMCC7326 mutant strain msn4 was used for all the built Y. lipolytica transformant strains.The K. marxianus CGMCC2.1977strain was used for all the built K. marxianus transformant strains.P. pastoris GS115 was used for all the built P. pastoris transformant strains.Yeast strains were grown at 30 °C in a YPD medium (10 g/L yeast extract, 5 g/L tryptone, and 20 g/L glucose).When necessary, transformants were screened by adding hygromycin to the YPD.E. coli DH5α was used for the amplification of plasmids.E. coli BL21 (DE3) was used for plasmid expression.The E. coli strains were cultivated at 37 °C in a Luria-Bertani medium (LB) supplemented with ampicillin (100 mg/L) or kanamycin sulfate (50 mg/L).C. glutamicum ATCC13032 was grown in LBHIS medium (LB supplemented with brain heart infusion and sorbitol: 5 g/L tryptone, 2.5 g/L yeast extract, 18.5 g/L brain heart infusion broth, 91 g/L sorbitol and 5 g/L NaCl) at 30 °C with chloramphenicol (10 μg/mL) to screen transformants.For solid media, agar (15 g/L) was added.

General molecular biology methods
Restriction endonucleases and DNA polymerases were purchased from Thermo Fisher Scientific.High fidelity Taq DNA polymerase (KOD plus, Toyobo) was used for DNA cloning.ExTaq DNA polymerase (Takara) was used for genotype verification.The PCR-amplified products in the agarose gels were purified using a GeneJet Gel Extraction Kit (Thermo Scientific).PCR-amplified products were subcloned into a vector using EasyFusion Assembly Master Mix (New Cell & Molecular Biotech, Suzhou, China).Genewiz (Suzhou, China) performed the primers synthesis.

Plasmid construction
We selected five constitutive promoters from Y. lipolytica and from K. marxianus respectively (Table 2) for identification of promoter expression levels in Y. lipolytica, K. marxianus, P. pastoris, E. coli and C. glutamicum.All plasmids, comprising each promoter and the α-amylase (Oryza sativa, AMY1A, 1305 bp), or the RFP gene (mRuby, JX489389.1,771 bp) as reporter genes, were derived from the skeletal plasmid pSWV-hph (Fig. S1).The plasmid pSWV-hph contains parts of 26S rDNA for integration, hp4d promoter, aep terminator, hygromycin resistance gene (hph) and ampicillin resistance gene (amp r ) [49] and was obtained from laboratory storage.The plasmid schematic is shown in Fig. S1.The 26S rDNA sequences in shuttle expression vectors are homologous across various yeast strains [50,51].The putative promoter regions were amplified by PCR using the primers shown in Table S2 and the genomic Y. lipolytica DNA or K. marxianus DNA as templates.The promoter sequences and the reporter genes, α-amylase and RFP, are listed in the supplemental material.Detailed information for constructing the plasmids in this study is listed in Table S1.The primers for verifying the constructed plasmids are listed in Table S2.

The non-conventional yeasts and bacterium transformation
Transformant strain details used in this study are shown in Table 3.The PCR products of the constructed promoter-reporter plasmids, with a pair of primers, Broadhost vector-F/ Broad-host vector-R (Table S2), were purified from the agarose gel.Additionally, they were used to transform yeast strains Y. lipolytica msn4, K. marxianus CGMCC2.1977 and P. pastoris GS115.Yeasts were transformed using the lithium acetate method described by Chen et al. [52].
The yeast strain taken from -70 °C was spread on a YPD plate and incubated at 30 °C for 20 h.The cells were scraped from the surface of the plate and added into a sterile 1.5 mL microcentrifuge tube.In the microcentrifuge tube, cells were in the presence of 82 μL polyethylene glycol 4000 (50%, w/v), 5 μL 2 M dithiothreitol, 3.5 μL 3 M lithium acetate, 5 μL 5.0 mg/mL single-stranded carrier DNA (heated in a boiling water bath for 5 min and then chilled in ice/water) and 5 μL linearized DNA (about 1 μg/μL).The transformation mix was thoroughly vortexed.The tube was incubated at 39 °C for 60 min and then centrifuged at 2000 rpm at room temperature for 5 min.The supernatant was discarded and 500 μL YPD medium was added to suspend the cells.The cells were recovered at 30 °C for 60 min and spread directly on a well-dried selective plate and incubated at 30 °C.The transformant colonies appeared about 48 h after transformation and the colonies were picked and verified with corresponding validation primers (Table S2).
E. coli and C. glutamicum were transformed using the methods by Hu et al. [53].Overnight, the E. coli culture was inoculated into 50 mL LB media at 37 °C and 200 rpm until OD 600 reached 0.5.The E. coli cells were cooled on ice for 10 min, centrifuged, washed 3 times with ice-cold 0.1 M CaCl 2 , and stored at −70 °C in 1.5 mL aliquots.For transformation, an aliquot of competent cells was thawed on ice and 1-2 μL plasmid was added.The mixture was incubated on ice for 30 min and put in a 42 °C water-bath for 90 s.The mixture was then cooled on ice for 3 min and 400 μL LB media was added.The mixture was incubated at 37 °C and 200 rpm for 1 h and plated on LB agar containing antibiotics for selection.
Overnight, the C. glutamicum culture was inoculated into 40 mL of the Epo media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 30 g/L glycine, 1 g/L Tween-80) to  an initial OD 600 of 0.2.The culture was grown at 200 rpm and 30 °C until OD 600 reached 0.6.The cells were cooled on ice for 15 min, centrifuged, washed 3 times with icecold 10% glycerol, and stored at −70 °C in 1.5 mL aliquots.For electro-transformation, an aliquot of the competent cells was thawed on ice and 5 μL plasmid was added.The mixture was transferred to a cold electroporation cuvette (0.1 cm) and electroporated at 1.8 kV with a 5 ms pulse.Immediately after the electroporation, 1 mL LBHIS (5 g/L tryptone, 5 g/L NaCl, 2.5 g/L yeast extract, 18.5 g/L brain heart infusion powder and 91 g/L sorbitol) was added to the cuvette.The mixture was transferred to a 1.5 mL Eppendorf tube, incubated at 30 °C and 200 rpm for 1 h, and plated on LBHIS agar containing antibiotics for selection.

Relative amylase activities with starch-iodine assay
In Fig. 2a, the transformant strains of Y. lipolytica and K. marxianus with amylase under control of each promoter were spotted on YPD starch agar media (1% soluble starch) and incubated at 30 °C for 3 days or 6 days.The Y. lipolytica msn4 and K. marxianus strain CGMCC2.1977were used as the negative controls.Next, the plates were sprayed with an iodine solution.The iodine solution consisted of 25 g iodine into a saturated solution of 10 g potassium in 10 mL distilled water.The solution was stirred and dissolved, then added 500 mL ethanol and added distilled water to 1000 mL.Positive activity was defined as a clear halo around the colony on a purple background.From each transformant strain, 24 colonies were selected, spotted on YPD starch agar, supplemented with hygromycin, and incubated at 30 °C for 3 days or 6 days.Three colonies with the largest clear halos of each transformant strain were selected.The suspensions (100 µL) of serial dilutions (10 -4 times) of each colony were spread on YPD starch agar media to obtain isolates that stably expressed amylase.Next, the isolates were point inoculated on YPD starch agar media.The transformant strains with relatively strong amylase expression were cultured for 3 days.Strains with weak expression were cultured for 6 days and the three colonies of the transformant strainYL-km.FBA1-amy were used as the reference under both culture conditions (Fig. 2a).The diameters of the clear zones over the diameters of the colonies were measured using a ruler.Relative amylase activities of different transformant strains were compared by the Halo: colony ratio [54] (Table 4, Fig. 2b).In Fig. S2, the preliminary starch-iodine assays for different control strains were detected.

Absolute amylase activities with 3, 5-dinitrosalicylic acid (DNS) reducing sugar assay
The DNS method was used to determine the absolute amylase activity of each transformant strain.For each transformant strain, the three isolates with the highest Halo: colony ratios were chosen and cultured in 5 mL of YPG medium (10 g/L yeast extract, 5 g/L tryptone and 20 g/L glycerol) in a 50 mL tube at 30 °C for three days with rotary shaking at 220 rpm.The clear supernatants (crude extracellular amylase extracts) were obtained after centrifugation at 7800 × g for 10 min at 4 ºC two times.
A reaction mixture of 150 μL crude amylase extract and 300 μL 1% soluble starch solution was incubated in 0.1 M sodium phosphate buffer (pH 7.0) at 45 °C for 60 min.Subsequently, 600 μL DNS solution was added and boiled for 10 min for color development.The absorbance of the mixture was measured at 540 nm and compared to a prepared blank control solution (distilled water instead of crude amylase extract).The glucose concentration of each sample solution and the control solution was obtained from the glucose standard curve.The standard curve was made using 150 μL D-glucose (0.15 mg/mL; 0.3 mg/mL; 0.5 mg/mL; 0.7 mg/mL; 0.9 mg/mL; 1 mg/ mL).The glucose content of the sample was subtracted by the glucose content of the control.One unit of the amylase activity was defined as the amount of enzyme required to produce 1 µmol of reducing sugar under the assay conditions described [55] (Fig. 2c).The 540 nm absorbance of five-time diluted DNS reaction mixture of the controls for preliminary amylase activity assays were shown in table S3.

Quantitative fluorescence measurement and microscopic observation
For quantitative fluorescence measurement, five colonies of each yeast transformant strain with RFP under control of each promoter were cultured in 5 mL of YPD medium in a 50 mL tube at 30 °C for three days.One colony for each E. coli transformant strain with RFP under control of each promoter was cultured in 5 mL of LB medium for one day, two days, and three days, at 37 °C.One colony for each C. glutamicum transformant strain with RFP under control of each promoter was cultured in 5 mL of LBHIS medium for four days at 30 °C with rotary shaking at 220 rpm.Optical density of cultures, at a wavelength of 600 nm (OD 600 ), was measured with an UV-7504 spectrophotometer after dilutions to monitor cell growth.The value of OD 600 for each colony was measured and diluted to 1.The fluorescence intensity of 1OD for each colony was measured by a multifunctional microplate reader (Spark, TECAN) with monochromator settings as Ex 559 nm/Em 600 nm.The fluorescence intensities of 1OD different control strains for preliminary RFP quantitative fluorescence experiments were showed in table S4.In Fig. 3a, the fluorescence intensity of 1OD for K. marxianus strain CGMCC2.1977 was used as the negative control.In Fig. 4c, the fluorescence intensity of 1OD for E. coli BL21(DE3) was used as the negative control.In Fig. 6a, the fluorescence intensity of 1OD for P. pastoris GS115 and the fluorescence intensity of 1OD for C. glutamicum ATCC13032 were used as negative controls for P. pastoris transformants and C. glutamicum transformants, respectively.
For microscopic observation, among the five colonies of each yeast transformant strain, the one with the highest fluorescence value was selected and cultured in YPD medium.One colony for each E. coli transformant strain was cultured in LB medium and one colony for each C. glutamicum transformant strain was cultured in LBHIS medium.Confocal images were collected using a confocal microscope (Ti-E Nikon A1R HD25, Tokyo, Japan).In Fig. S3, microscopic RFP fluorescence images of the different control strains were detected.

Strategy for the screening of broad-host expression promoters for construction of broad-host expression vectors
Our objective was to evaluate a wide range of promoter sets with varying strengths to create versatile expression vectors and shuttle plasmids to effectively regulate the metabolic flow for the optimal synthesis of valuable bioproducts in various organisms, including non-conventional yeasts (Y.lipolytica, K. marxianus, P. pastoris) and bacteria (E.coli, C. glutamicum).

Amylase expression under each promoter in Y. lipolytica and K. marxianus
We used α-amylase as a reporter gene to examine the expression strengths of the ten promoters in Y. lipolytica and K. marxianus.The amylase activity is the ability to degrade starch and is easy to measure (see "Materials and methods" section).Thus, amylase is a good candidate for examining the relationship between gene expression and promoter strength.The PCR products of the ten promoter-amylase plasmids were used to transform the non-conventional yeasts Y. lipolytica msn4 and K. marxianus CGMCC2.1977 to yield twenty transformant strains (Table 3).We used two methods for amylase activity assays: a starch-iodine assay for relative amylase activities and DNS reducing sugar assay for absolute amylase activities.
The starch-iodine assay is useful for rapid screening on the transformants of large populations with high or low amylase activities.Positive amylase active colonies were surrounded by a bright orange halo on YPD starch agar media by spraying iodine solution [54].Genomic integration mediated by 26S rDNA will cause differences in integration sites and copy numbers, which caused the amylase expression levels for the transformant strain colonies to vary.Despite colony variations, the mean expression levels of the colonies can be used for a rough estimation of expression levels [57].The three isolates of each transformant strain with the largest clear halos were selected and cultured on YPD starch agar media for 3 or 6 days.The transformant strain, YL-km.FBA1-amy, was used as the reference under both culture conditions (3 or 6 days) (Fig. 2a).The relative expression strength of amylase, under control of each promoter in both Y. lipolytica and K. marxianus, were compared by the Halo: colony ratio (Fig. 2b and Table 4).
We observed that the amylase activities varied with promoter strength in different transformant strains.In the category of the five Y. lipolytica recombinant strains expressing amylase via its native promoters, the strains containing yl.TEF1, yl.TDH1 and yl.EXP1 had strong expression strengths with the mean Halo:Colony ratios (3.93, 3.83, 4.04 respectively).The strain containing yl.hp4d had relatively weaker expression strength with the mean Halo: colony ratio 3.22.The strain containing yl.FBA1in had the weakest expression strength with the mean Halo:Colony ratio 2.81.The relative strength is as follows: yl.TEF1 ∼ yl.TDH1 ∼ yl.EXP1 > yl.hp4d > yl.FBA1in.In the category of the five K. marxianus recombinant strains expressing amylase via its native promoters, the strains containing km.PDC1 and km.TEF1 had relatively strong expression strengths with the mean Halo: colony ratios (3.38 and 3.05 respectively).The other three strains containing km.FBA1, km.TDH3 and km.ENO1 had relatively weaker expression strengths with the mean Halo:Colony ratios (2.98, 2.48, 2.81 respectively).The relative strength is as follows: km.PDC1 ∼ km.TEF1 > km.FBA1∼ km.TDH3 ∼ km.ENO1.
In the category of the five Y. lipolytica recombinant strains via K. marxianus promoters, the Y. lipolytica strains containing km.TDH3, km.ENO1, km.PDC1 and km.FBA1 had strong expression strengths with the mean Halo: colony ratios (2.61, 2.89, 2.48 and 2.21 respectively), which were similar to the ratio of the Y. lipolytica strain containing yl.FBA1in.The Y. lipolytica strain containing km.TEF1 had very weak expression strength with the mean Halo: colony ratio 1.57.The relative strength is as follows: km.TDH3 ∼ km.ENO1 ∼ km.PDC1∼ km.FBA1 > > km.TEF1.In the category of the five K. marxianus recombinant strains via Y. lipolytica promoters, the K. marxianus strains containing yl.TEF1 and yl.TDH1 had strong expression strengths with the mean Halo: colony ratios (2.76 and 2.71 respectively), which resembled the K. marxianus strains containing km.FBA1, km.TDH3 and km.ENO1.The K. marxianus strains containing yl. hp4d and yl.EXP1 showed very low expression with the mean Halo: colony ratios (1.29 and 1.37 respectively).The K. marxianus strain containing yl.FBA1in in particular couldn't detect clear halos around the colonies.The relative strength is as follows: yl.TEF1 ∼ yl.TDH1 > > yl.hp4d ∼ yl.EXP1 > yl.FBA1in.
Five replicates of each isolate with the highest Halo:Colony ratio for each transformant strain were cultured in YPG medium for three days with rotary shaking for absolute amylase activity quantification in liquid cultures using the DNS method (see "Materials and methods" section) [55].The results of the DNS reducing sugar assay (Table 4 and Fig. 2c) aligned with the Halo:Colony ratio results of starch-iodine assay (Table 4 and Fig. 2b) with only slight differences.This may be the results of the starch-iodine assay were the average value of amylase activities expressed by three different colonies of each transformant strain.In Fig. 2c, the Y. lipolytica strains expressing amylase via its native promoters showed high amylase activities ranging from 38.02 U/mL to 41.99 U/ mL.The K. marxianus strains expressing amylase via its native promoters also showed high amylase activities ranging from 36.67 U/mL to 42.46 U/mL.The Y. lipolytica strains containing km.PDC1, km.FBA1, km.TDH3, km.ENO1 and the K. marxianus strains containing yl.TEF1, yl.TDH1 had high amylase activities (37.73 U/ mL, 40.78 U/mL, 42.55 U/mL, 44.36 U/mL, 43.65 U/ mL, 46.29 U/mL respectively), which resembled the Y. lipolytica strains and the K. marxianus strains expressing amylase via their native promoters.The Y. lipolytica strains containing km.TEF1 and the K. marxianus strains containing yl.hp4d, yl.FBA1in, yl.EXP1 showed very weak amylase expression with very low amylase activities at 17.29 U/mL, 5.71 U/mL, 2.57 U/mL and 19.6 U/ mL respectively.
The results showed that the five K. marxianus promoters in Y. lipolytica and the five Y. lipolytica promoters in K. marxianus can all express α-amylase with variable expression strength.The promoters km.PDC1, km.FBA1, km.TDH3, km.ENO1, yl.TEF1, yl.TDH1, highly express amylase in both Y. lipolytica and K. marxianus, can be used as the broad-spectrum promoters for construction of broad-host expression vectors to express heterologous synthesis pathways in different hosts and to assess appropriate expression chassis.The weak amylase expression promoters, km.TEF1 in Y. lipolytica, yl.hp4d, yl.FBA1in and yl.EXP1 in K. marxianus, can be used to express the metabolic flow essential for host growth and competitive for the heterologous metabolic pathway.

RFP expression under each promoter in Y. lipolytica and K. marxianus
We used RFP gene as the reporter gene to examine how the RFP expression varied with the strengths of the ten promoters in Y. lipolytica and K. marxianus.The RFP fluorescence is easy to detect and quantify in different hosts.We used two methods to check RFP gene expression.The fluorescence intensity for each transformant strain was measured by a multifunctional microplate reader to quantify RFP gene expression levels.Confocal images were collected using a confocal microscope for visual and qualitative view of RFP gene expression.
For quantitative fluorescence measurement, the 1OD fluorescence intensities (RFU) for five colonies of each transformant strain were measured by a multifunctional microplate reader with PMT (photomultiplier tube) gain value 80 (Table 5 and Fig. 3a).The fluorescence intensity of 1OD for K. marxianus strain CGMCC2.1977 was used as the negative control.The relative fluorescence intensities of the samples were subtracted by the fluorescence intensity of the control.The five Y. lipolytica recombinant strains expressing RFP via its native promoters showed drastically high fluorescence intensities compared to the other three categories.The strain containing yl.EXP1 had the highest RFP expression with the mean fluorescence intensity 4901.Among the five colonies of each transformant strain, the one with the highest fluorescence value was selected and cultured in YPD medium for one day, two days and three days for confocal microscopy.The confocal images for these transformant strains are shown in Fig. 3b.These transformant strains showed red fluorescence in cytosol and the red fluorescence brightness was different among the promoters used.The red fluorescence brightness of the confocal image for each transformant strain increased from the first day to the third day.In the category of the five Y. lipolytica recombinant strains expressing RFP via its native promoters, the red fluorescence brightness of the strains containing yl.hp4d, yl.TDH1 and yl.EXP1 were very high on the first day and the red fluorescence brightness of the strains containing yl.FBA1in and yl.TEF1 were relatively weak.In the category of the five K. marxianus recombinant strains expressing RFP via its native promoters, the red fluorescence brightness of the strains containing km.TEF1, km.TDH3 and km.ENO1 were highest.The red fluorescence of the strains containing km.PDC1 and km.FBA1 were never bright.The red fluorescence of the category of the five Y. lipolytica recombinant strains expressing RFP via K. marxianus promoters had the lowest brightness compared to the other three categories.In the category of the five K. marxianus recombinant strains expressing RFP via Y. lipolytica promoters, the high red fluorescence brightness of the K. marxianus strains containing yl.TEF1 and yl.TDH1 were comparable to the K. marxianus strains containing km.TEF1, km.TDH3 and km.ENO1.The K. marxianus strains The results showed that the five K. marxianus promoters all can express RFP in Y. lipolytica and the five Y. lipolytica promoters also all can express RFP in K. marxianus with variable expression strength.In our study, the five K. marxianus promoters km.PDC1, km.FBA1, km.TEF1, km.TDH3 and km.ENO1 did not highly express RFP in Y. lipolytica and did not coordinate with α-amylase expression.The Y. lipolytica promoters yl.TEF1 and yl.TDH1 have the potential to highly express amylase and RFP in K. marxianus.The K. marxianus promoters km.PDC1, km.FBA1, km.TDH3 and km.ENO1 only have the potential to highly express amylase in Y. lipolytica.The Y. lipolytica promoters yl.hp4d, yl.FBA1in, and yl.EXP1 could weakly express RFP in K. marxianus, which coordinates with α-amylase expression.Our results revealed that the correlation between α-amylase expression and RFP expression in each transformant strain was weak.These results underscore that gene expression is not always linearly related to promoter strength, which may vary and depend on the specific gene.
In most cases, gene expression and activity were correlated with promoter strength [29,35,59].However, the stronger promoters are not always better for expressing different exogenous genes.For example, the strong T7 native promoter was also not always better for expressing different exogenous genes in E. coli.In our results (Sect."Yeast shuttle vectors expressed in Escherichia coli"), T7 promoter could strongly express RFP (Fig. 4), but could only weakly express amylase (Fig. 5) in E. coli.The different expression levels of RFP and α-amylase under the control of T7 promoter in E. coli further verified that the gene expression level by the same promoter depends on the specific gene.

Yeast shuttle vectors expressed in Escherichia coli
We discovered that the km.TEF1 promoter from K. marxianus could be used for shuttle expression in E. coli.During the cloning of plasmid pkm.TEF1-rfp (Table S1) in E. coli DH5α, we observed that the colonies of the E. coli DH5α transformant strain containing plasmid pkm.TEF1-rfp would turn red in color.The finding suggests that the eukaryotic km.TEF1 promoter from K. marxianus has the ability to allow gene expression in the prokaryotic host E. coli.A similar study reports that the eukaryotic promoter GAL1/10 from S. cerevisiae could directly express genes in the E. coli [46].Any piece of random DNA unlikely to be a functional promoter is not that far from a functional bacterial promoter.A single mutation for each of the evolved random sequences was found to confer the promoter function and can be further increased in a stepwise manner by additional mutations that improve similarity to canonical promoters in E. coli [60].So, the km.TEF1 promoter from K. marxianus may happen to have similar elements to bacterial promoters.In Fig. 4a, we used the well characterized bacteriophage T7 promoter sequence as a reference for comparison with the eukaryotic km.TEF1 promoter sequence from K. marxianus.The −34 to −18 positions of the yeast km.TEF1 promoter sequence has similarity with the T7 promoter, suggesting a common promoter function of this region as T7 promoter.The recognition region (positions −17 to −5) of the T7 native promoter includes the AT-rich recognition loop (positions −17 to −13) and the specificity loop (positions −12 to −5).These provide a sequence-specific recognition by the bacteriophage T7 RNA-polymerase (RNAP) [61].The T7 RNAP can recognize the sequences closely related to the T7 native promoter [62].The km.TEF1 promoter shares the similar sequences of the AT-rich recognition loop (positions −34 to −30), the specificity loop (positions −29 to −22) and the bacteriophage core region (positions −25 to −18) with T7 promoter.
To further characterize the behavior of the km.TEF1 promoter in E. coli, we also transformed E. coli BL21 (DE3) with plasmid pkm.TEF1-rfp to yield the transformant strain DE3-km.TEF1-rfp, and we transformed E. coli DE3 with plasmid pET28a-rfp to yield the transformant strain DE3-pET28a-rfp for comparing to the most studied T7 expression system.The skeletal plasmid pSWV-hph [63] used for constructing the plasmid pkm.TEF1-gene and the pET28a used for constructing the plasmid pET28a-gene with the same origin PBR322 of E. coli are high-copy-number plasmid [64].In Fig. 4b, the transformant strains DE3-pET28a-rfp, DE3-km.TEF1-rfp and DH5α-km.TEF1-rfp were spotted on the LB agar medium with or without 100 μM IPTG for two days, three days, and four days.Obvious red color of the strain DE3-pET28a-rfp was observed when induced by IPTG, while the strain DE3-pET28a-rfp without IPTG was white and vaguely red.The strains DE3-km.TEF1rfp and DH5α-km.TEF1-rfp with or without IPTG all showed obvious red color and red color darkened with the increase of days.Only the strain DE3-km.TEF1rfp would differentiate into some white color colonies.Confocal images of the white colonies and red colonies showed that there was no red fluorescence in the white colonies.Additionally, some of the red colonies no longer expressed RFP.This may be because the native plasmid of E. coli DE3 cannot coexist with the plasmid pkm.TEF1rfp, leading to the loss of exogenous plasmids with the prolongation of growth time.
The transformant strains DE3-pET28a-rfp, DE3-km.TEF1-rfp, and DH5α-km.TEF1-rfp were also cultured in liquid LB medium with or without 100 μM IPTG for one day, two days and three days to measure fluorescence intensity (Fig. 4c) and confocal microscopy (Fig. 4d).In Fig. 4c, the fluorescence intensity of 1 OD 600 each transformant strain was measured with PMT gain value 70.The T7 promoter was so strong that RFP fluorescence exceeded the measurable range.Therefore, we lowered the gain value from 80 to 70.The fluorescence intensity of each transformant strain for one day, two days, and three days became higher.The difference between the second day and the third day was not evident.DE3-pET28arfp with RFP under control of the T7 promoter had the strongest fluorescence intensity (more than 20,000 RFU on the second day) under the induction of IPTG.RFP expression of the strain DE3-pET28a-rfp without IPTG induction was the weakest (about 2000 RFU on the second day).The promoter km.TEF1 can express RFP in both E. coli DE3 and E. coli DH5α.The fluorescence intensities of the strains DE3-km.TEF1-rfp and DH5αkm.TEF1-rfp with or without IPTG were about 4000 or 5000 RFU with no significant difference among them.This indicates that the promoter, km.TEF1, should be classified as a strong constitutive promoter in E. coli.In Fig. 4d, the confocal images showed that these transformant strains had strong RFP expression and the red fluorescence was already very high on the first day.
We transformed the plasmids, pyl.TEF1-amy and pkm.TEF1-amy, with amylase under control of the promoters yl.TEF1 and km.TEF1, respectively, into E. coli DE3 and DH5α to further confirm whether the yeast promoters could be used to drive gene expression in E. coli.We transformed E. coli DE3 with plasmid pET28a-amy to be able to compare the T7 expression system.In Fig. 5a, the transformant strains DE3-pET28a-amy, DE3-pyl.TEF1-amy, DH5α-pyl.TEF1-amy, DE3-km.TEF1-amy, and DH5α-km.TEF1-amy were spotted on LB starch agar medium with or without 100 μM IPTG for four days.Then the plates were sprayed with iodine solution.The strain DE3-pET28a-amy with amylase under control of the T7 promoter had a small clear halo around the colony under the induction of IPTG and had no clear halo without IPTG induction.This indicates that the T7 promoter could not express amylase well in E. coli.The strain DE3yl.TEF1-amy with or without IPTG had no clear halo, indicating that the promoter yl.TEF1 failed to express amylase in E. coli DE3.The strain DH5α-yl.TEF1-amy with or without IPTG had clear halos around the colonies, indicating that the promoter yl.TEF1 could express amylase in E. coli DH5α.The strains DE3-km.TEF1-amy and DH5α-km.TEF1-amy with or without IPTG all had clear halos around the colonies, indicating that the promoter km.TEF1 could express amylase in both DE3 and DH5α.The clear halos around the colonies of the strains DH5α-yl.TEF1-amy, DE3-km.TEF1-amy and DH5α-km.TEF1-amy with or without IPTG were approximately the same size and larger than the strain DE3-pET28aamy with IPTG.The Halo:Colony ratio for each transformant strain was measured to quantify amylase activity (Fig. 5b).The results of the RFP expression strength showed that the promoter km.TEF1 could reach ~ 20% of the T7 promoter in E. coli.The expression of RFP and α-amylase by the promoter km.TEF1 in E. coli was not affected by the inducer IPTG.This indicated that the promoter km.TEF1 is a constitutive promoter in E. coli.The expression of α-amylase by the promoter yl.TEF1 in E. coli was also not affected by the inducer IPTG, indicating that the promoter yl.TEF1 is also a constitutive promoter in E. coli.The α-amylase expression was not high in E. coli under the control of T7 promoter, yl.TEF1 promoter and km.TEF1 promoter.The different expression levels of RFP and α-amylase under the control of T7 promoter or km.TEF1 promoter in E. coli further verified that the gene expression level by the same promoter depends on the specific gene.
We selected five colonies from each of the two P. pastoris transformant strains, PP-yl.TEF1-rfp and PP-km.TEF1-rfp, cultured in YPD medium for three days with rotary shaking.One colony from each of the two C. glutamicum transformant strains CG-yl.TEF1-rfp and CG-km.TEF1-rfp was selected and cultured in LBHIS medium for four days with rotary shaking.In Fig. 6a, the 1 OD 600 fluorescence intensity of each transformant strain was measured with gain value 80.The strain PP-yl.TEF1-rfp containing yl.TEF1 and the strain PP-km.TEF1rfp containing km.TEF1 could express RFP in P. pastoris with the mean fluorescence intensities 131.1 and 40.3 RFU, respectively.The strain CG-km.TEF1-rfp containing km.TEF1 had the highest RFP expression level (970.3RFU).The strain CG-yl.TEF1-rfp had the weakest RFP expression level (20.7 RFU).In Fig. 6b, the one had the highest fluorescence value for each of the two strains PP-yl.TEF1-rfp and PP-km.TEF1-rfp was selected for confocal microscopy.The strains PP-yl.TEF1-rfp and CG-km.TEF1-rfp had high red fluorescence brightness, while the strains PP-km.TEF1-rfp and CG-yl.TEF1-rfp had weak red fluorescence brightness.
The results showed that the Y. lipolytica promoter yl.TEF1 highly expressed α-amylase and RFP in yeast K. marxianus and in yeast P. pastoris.The K. marxianus promoter km.TEF1 highly expressed RFP in bacterium E. coli and in bacterium C. glutamicum.The red fluorescence in the bacterium C. glutamicum strain CG-km.TEF1-rfp was more than 5 times lower than the E. coli strains DE3-km.TEF1-rfp and DH5α-km.TEF1-rfp.The plasmid pkm.TEF1-rfp exhibited a higher level of red fluorescent protein expression in E. coli compared to the plasmid pXMJ19-km.TEF1-rfp in C. glutamicum.This suggests that although the km.TEF1 promoter was capable of expressing red fluorescent protein in both bacteria, its expression was more robust in E. coli.It is not surprising that these promoters function across yeasts and bacteria, since yeast promoters tend to be transferable across yeasts within a certain genetic distance [67,68] and that bacterial promoters could possibly exist by chance within yeast promoters.It is still useful that these particular sequences were found to function across hosts.Long nucleosome free regions (NFR) in promoters were evolutionarily conserved.The conserved NFR sequences included the transcription factor binding sites and multiple stretches of poly-A or poly-T.This may be one explanation for some promoters functioning across hosts [69].

Conclusion
The development of broad-spectrum promoter libraries comprising promoters of varying strengths for different hosts are attractive and meaningful to biosynthetic engineers.As there is no pattern to what promoters will be active in another host.There is also unpredictability when using different genes of interest.So, for gene expression, a large number of different promoters need to be screened.In this study, we found that the five K. marxianus promoters in Y. lipolytica and the five Y. lipolytica promoters in K. marxianus could all express α-amylase and RFP with variable expression strength.In addition, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability by promoting gene expression in P. pastoris, E. coli, and C. glutamicum.It is worth mentioning that the yeast P. pastoris displayed strong expression of amylase and RFP in response to the yl.TEF1 promoter.On the other hand, both E. coli and C. glutamicum bacteria exhibited robust synthesis of RFP in response to the eukaryotic km.TEF1 promoter.It is interesting that the RFP gene expression level of the km.TEF1 promoter reached 20% of the T7 promoter in E. coli.These results suggest that actively controlled strategies to optimize carbon flow and enhance bioproduct synthesis in numerous microbial species are possibly feasible by the distinctive capabilities of non-conventional yeast promoters.Notwithstanding these pioneering discoveries, the research acknowledges specific constraints.Only two visible reporter genes (α-amylase and RFP) were tested.The gene expression level was not always correlated with promoter strength and depends on the specific gene.The reliabilities of these promoters across hosts need to be further verified with additional reporter genes.Through the novel implementation of broad-spectrum promoters, this study has the capacity to significantly advance the development of adaptable, dynamically controlled systems in different hosts.These promoters, having the broad-host range expression potentials, might improve bioproduction efficiency and versatility by optimally controlling pathways of engineering.

Fig. 3 Fig. 4 Fig. 5
Fig. 3 Expression analysis of RFP in Y. lipolytica and K. marxianus transformant strains.a Mean RFP fluorescence intensities (RFU) of the colonies (n = 5) of each Y. lipolytica or K. marxianus transformant strain with reporter gene RFP under control of each promoter cultured for three days are shown with standard errors (gain value 80); b Microscopic fluorescence images of the colony with the highest fluorescence value of each Y. lipolytica or K. marxianus transformant strain cultured for one day, two days, three days.Fluorescent images of the strains were taken in the same setting

Fig.
Fig. Yeast Shuttle Vectors Express RFP and α-amylase in P. pastoris and C. glutamicum.a RFP fluorescence of each transformant strain cultured for three days was measured in three wells in a 96-well plate.Five colonies were selected for each P. pastoris transformant strain and one colony was selected for each C. glutamicum transformant strain.The means and standard deviations were shown (gain value 80); b Microscopic fluorescence images of each transformant strain cultured for three days; c Positive amylase activities detected by the clear halos around the colonies of starch-iodine assay

Table 1
Strains used in this study CGMCC: China General Microbiological Culture Collection Center; ATCC: America Type Culture Collection

Table 2
List of promoters used in this study The elements used in this study are listed with their names, open reading frames regulated, accession numbers, and base pair ranges

Table 3
Transformant strains used in this study

Table 4
Amylase activities produced by the transformant strains with amylase under control of each promoter a Three colonies for each transformant strain; b Five replicates of the colony with the largest halo:colony ratio for each transformant strain KM-yl.TEF1-amy 2.76 ± 0.15 43.65 ± 1.77 KM-yl.TDH1-amy 2.71 ± 0.52 46.29 ± 1.53 KM-yl.EXP1-amy 1.37 ± 0.06 25.36 ± 2.10 2 RFU.The strains containing yl. hp4d and yl.TDH1 had relatively weaker RFP expression with the mean fluorescence intensities of 2671.1 RFU and 2795.2RFU, respectively.The strains containing yl.FBA1in and yl.TEF1 had the weakest RFP expressions with the mean fluorescence intensities 1358.5 RFU and 1162.8RFU, respectively.The relative strength is as follows: yl.EXP1 > > yl.TDH1 ∼yl.hp4d > > yl.FBA1in ∼ yl.TEF1.In the category of the K. marxianus recombinant strains expressing RFP via its native promoters, the strains containing km.TEF1, km.ENO1 and km.TDH3 had relatively strong RFP expression with the mean fluorescence intensities 444.7 RFU, 598.2 RFU, and 627.6 RFU, respectively.The strains containing km.PDC1 and km.FBA1 had relatively weak RFP expression with mean fluorescence intensities of 191.4 RFU and 221.0 RFU, respectively.The relative strength is as follows: km.ENO1 ∼ km.TDH3 > km.TEF1 > km.FBA1 ∼ km.PDC1.The five

Table 5
RFU of the transformant strains with reporter gene RFP under control of each promoter (Gain value 80) Y. lipolytica recombinant strains expressing RFP via K. marxianus promoters had extremely lower fluorescence intensities compared to the other three categories ranging from 3.8 RFU to 44.6 RFU.In the category of the five K. marxianus recombinant strains expressing RFP via Y. lipolytica promoters, the K. marxianus strains containing yl.TEF1 and yl.TDH1 showed strong fluorescence intensities at 351.5 RFU and 326.6 RFU, respectively.This was comparable to the fluorescence intensity of the K. marxianus strain containing km.TEF1 at 444.7 RFU.The K. marxianus strains containing yl.hp4d, yl.TDH1 and yl.EXP1 had weak expression strength with mean fluorescence intensities of 12.9 RFU, 14.8 RFU and 19.6 RFU, respectively.The relative strength is as follows: yl.TEF1 ∼ yl.TDH1 > > yl.hp4d ∼ yl.TDH1 ∼ yl.EXP1.